Central to the hormonal control of metabolism is the specific binding of insulin to the ectodomain of the insulin receptor. This application seeks to delineate the mechanism of such binding. The proposed studies will characterize insulin's active conformation, define points of contact between insulin and the receptor, and test functional models of the hormone-holoreceptor complex. Collaborative co-crystallographic studies of a specific complex between insulin and a monomeric minimized fragment of the receptor alpha subunit will be undertaken with G. G. Dodson (University of York, England). The following propositions will be tested: Hypothesis 1. That insulin's active conformation contains a combination of induced and preformed recognition elements; Hypothesis 2. That insulin's active conformation contains two discrete recognition surfaces in separate contact with the two alpha subunits of the alpha2beta2 holoreceptor; and Hypothesis 3. That wild-type and variant co-crystal structures of a specific complex between the hormone and receptor will rationalize the large existing body of mutagenesis data and facilitate design of novel ligands. Aim 1 focuses on NMR and crystallographic studies of non-standard insulin analogues, thus continuing the objectives of the previous application. Aims 2-4 are new. In Aim 2, we propose to obtain a map of hormone-receptor contacts by systematic analysis of photo-cross-links between insulin derivatives and the receptor; derivatives will be prepared by P. G. Katsoyannis (Mt. Sinai Medical Center, NY, NY). Novel "bifunctional" insulin derivatives will be prepared to test hypothesis 2. In Aim 3, we propose to purify alpha subunit fragments for co-crystallization trials with native and variant insulins. Design of such fragments will be undertaken in collaboration with J. Bass (Northwestern University). Aim 4 focuses on co-crystallization studies of the hormone-receptor complex with the Dodson laboratory. Each aim is supported by a solid foundation of preliminary results.